nous enzyme by free recombinant species, to assess the contribution of that ... this domain binds to another component of the complex, but might primarily serve a targeting function ... extension of rat or human AspRS [6, 71, the N-terminal 200-.
Eur. J. Biochem. 243, 259-267 (1997) 0 FEBS 1997
Aspartyl-tRNA synthetase from rat In vitro functional analysis of its assembly into the multisynthetase complex Fabrice AGOU and Marc MIRANDE Laboratoire d'Enzymologie et Biochirnie Structurales, Centre National de la Recherche Scicntifique, Gif-sur-Yvette,France (Received 12 August 1996) - EJB 96 1202/2
In mammalian cells, nine aminoacyl-tRNA synthetases, including aspartyl-tRNA synthetase, are associated within a multienzyme complex. Rat aspartyl-tRNA synthetase has a N-terminal polypeptide extension of about 40 amino acid residues which can be removed without impairing its catalytic activity. Earlier, in vivo studies showed that enzymes deprive of this N-terminal segment behave in vivo as free entities. We designed an experimental in vitro approach, based on the exchange of the complexed endogenous enzyme by free recombinant species, to assess the contribution of that domain in the association of aspartyl-tRNA synthetase to the complex. A phosphorylation site was introduced at the N-terminus of rat aspartyl-tRNA synthetase. The enzyme served as a reporter protein to evaluate the dissociation constants of native and N-terminal-truncated species towards the complex. Our data show that a moderate but significant drop in affinity is inferred by the removal of the N-terminal domain. The results suggest that this domain binds to another component of the complex, but might primarily serve a targeting function absolutely required in vivo for the assembly within the multienzyme structure. Keywords: complex assembly ; aminoacyl-tRNA-synthetase complex ; mammalian cells.
In higher eukaryotes, aminoacyl-tRNA synthetases are encountered as free enzymes or as components of high molecularmass complexes. Two different complexes have been described to date. One is formed by association of valyl-tRNA synthetase (ValRS) with the a, p, y and 8 subunits of elongation factor 1 11-31. One of the key elements for the interaction between ValRS and elongation factor 1 resides in the N-terminal polypeptide extension which characterizes mammalian ValRS [4] apart from its bacterial or yeast counterparts, enzymes that do not associate into large structures. The other example is a multienzyme complex comprising synthetases specific for glutamic acid, proline, isoleucine, leucine, methionine, glutamine, lysine, arginine and aspartic acid, as well as three additional components with apparent molecular masses of 43, 38 and 18 kDa [5]. Essentially identical complexes could be isolated from insects to mammals. A special issue concerns the mode of assembly of these enzymes into a well-defined and tightly organized structure. Earlier studies have shown that the N-terminal 34-amino-acid extension of rat or human AspRS [6, 71, the N-terminal 200amino-acid extension and the 75-amino-acid internal repeats of the multifunctional glutamyl .prolyl-tRNA (GluProRS) from fly or human 18, 91, the N-terminal 73-amino-acid extension of hamster and human arginyl-tRNA synthetase (ArgRS) [lo, 1 I], Correspondence to M. Mirande, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, F-91198 Gif-sur-Yvette Cedex, France Abbreviations. ArgRS, arginyl-tRNA synthetase; AspRS, aspartyltRNA synthetase; GlnRS, glutaminyl-tRNA synthetase ; GluProRS, the bifunctional enzyme glutamyl .prolyl-tRNA synthetase; IleRS, isoleucyl-tRNA synthetase; ValRS, valyl-tRNA synthetase; rAspRS, recombinant rat AspRS produced in yeast; rAspRSK,rAspRS with an engineered phosphorylation site; rAspRSK-P,phosphorylated form of rAspRS"; AspRS -AN20 and AspRS -AN36, rAspRS with N-terminal deletions of 20 or 36 residues, respectively. Enzyme. Aspartyl-tRNA synthetase (EC 6.1.1.12).
the two C-terminal repeated units of human isoleucyl-tRNA synthetase (IleRS) [ 121 and the N-terminal 250-amino-acid extension of human glutaminyl-tRNA synthetase (GlnRS) [13] are idiosyncratic features of the mammalian enzymes. Since ArgRS or AspRS behave in vivo as free enzymes after removal of their N-terminal extensions [ 14, 151, the polypeptide extensions that characterize mammalian synthetases are likely to be involved in complex assembly. In addition, a role in the transfer of the aminoacyl-tRNA from the synthetase to the a subunit of elongation factor 1 was also ascribed to this extension [16]. We previously shown that rat AspRS produced in yeast, as well as N-terminal truncated derivatives, are functionally indistinguishable in the aminoacylation reaction [17]. In the present paper, the possibility that these catalytically dispensable domains are composed of protein motifs responsible for association of the components of the multisynthetase complex has been tested experimentally.
EXPERIMENTAL PROCEDURES The native recombinant AspRS from rat (rAspRS), and the N-terminal truncated derivatives AspRS - AN20 or AspRS AN36, produced in yeast, were isolated as previously described [17]. Homogcneous yeast AspRS was a gift of D. Moras (IGBMC, Strasbourg, France). The multisynthetase complex from rat liver was purified according to [IS]. Enzyme assay. AspRS was assayed by the aminoacylation of tRNA, as previously described 1171. One unit of AspRS activity is the amount of enzyme producing 1 nmol aspartyl-tRNA/ min, at 25 "C. Specific activities of purified enzymes were calculated with protein concentrations determined using absorption coefficients of 0.41, 0.41, 0.38 and 0.39 AZ80units.mg ' .cm2
260
Agou and Mirande ( E m J. Biochern. 243)
for rAspRS, rAspRS with an engineered phosphorylation site (rAspRSK), AspRS - AN36 and AspRS -AN20, respectively. Construction of pYeDP60/DRSK.The BnmHI-XbaI fragment from M13mp9/DRS-AN20 [I71 was replaced by the BunzHI-XbaI linker L03, made of the two complementary oligonucleotides F03 (5’-GATCCATAATGCCCAGAAGAGCTTCCGTTT-3’) and F03-IC (3’-GTATTACGGGTCTTCTCGAAGGCAAAGATC-5’) to give MI 3mp9/DRSK-AN20. Underlined nucleotides encode a recognition sequence (RRASV) for the catalytic subunit of CAMP-dependent protein kinase. The three cohesive linkers L08, made of the two oligonucleotides F08 (5’-CTAGAAAGGGTCAGGAGAAGCCGCG-3’) and F08-IC (3’-TTTCCCAGTCCTCTTCGGCGCCCTCT-S’), LIO, made of F10 (5’-GGAGATCGTGGACGCGGCGGAAGATTATGC-3’) and F10-IC (3’-AGCACCTGCGCCGCCTTCTAATACGATTTC-S’), and L12, made of F12 (5’-TAAAGAGAGATATGGGGTC-3’) and F12-IC (3’-TCTCTATACCCCAGAGCT-S’), were inserted into MI 3mp9/DRSK-AN20 digested by XbuI and XhoI. The resulting plasmid M13mp9/DRSKwas digested with BamHI and EcoRI and cloned into pYeDP60 digested by BamHI and EcoRI, to give pYeDP60/DRSK. All constructions were confirmed by DNA sequencing. Expression and purification of rAspRSK produced in yeast. The yeast cell W303-IB (a) was transformed with pYeDP60/DRSK. The expression and purification of the recombinant enzyme rAspRSKwas conducted essentially as described for rAspRS [ 17 1. The addition of the pentapeptide RRASV modifies the global net charge of the protein to: +2 compared to the net charge of rAspRS. Accordingly, the chromatographic behavior of rAspRSK was slightly modified. The protein was eluted on S Sepharose FF, Q Sepharose FF and tRNA-Sepharose at phosphate concentrations of 130, 100 and 140 mM. The homogeneity of the enzyme preparation was checked by SDSPAGE conducted according to the method of Laemmli [19]. Phosphorylation of rAspRSK. Phosphorylation was conducted in a final volume of 0.15 ml containing 0.5 mg purified rAspRSK (0.03 mM), 80 units catalytic subunit of CAMP-dependent protein kinase from bovine heart (40000 units/mg; Sigma) and 0.5 mCi [y”P]ATP (0.1 mM, 30 Ci/mmol; Amersham) in 20 mM Tris/HCl, pH 7.5, 100 mM NaC1, 12 mM MgCI,, 1 mM 1,4-dithioerythritol and 20% glycerol. After incubation for 14 hours at 10”C, complete phosphorylation of rAspRSK was achieved by addition of ATP to 1 mM and further incubation at 10°C for 2 hours. The incubation mixture was applied to a Sephadex G25 column (0.6 cmX25 cm) equilibrated in 100 mM potassium phosphate, pH 7.5, 1 mM 1,4-dithioerythritol, 10% glycerol, and developed at 20°C at a flow rate of 0.2 ml/min to remove unincorporated ATP. The phosphorylated protein rAspRS“-P, eluted in the flow-through fractions, was dialyzed against 25 mM potassium phosphate, pH 7.5, 1 mM IA-dithioerythritol, 50% glycerol, and stored at -20°C. The radiolabelled protein has a specific activity of about 0.3 Ci/g. The ensuing phosphorylated protein was analyred on a Mono S HR 5/5 column (Pharmacia) to determine the extent of radiolabelling of the two sites of the dimeric cnzyme. The column, developed at 20°C at a flow rate of 0.8 ml/min, was equilibrated in 25 mM potassium phosphate, pH 7.5, 10 mM 2-mercaptoethanol, 20% glycerol, and elution was accomplished by a 30-ml linear gradient of potassium phosphate of 25-400 mM, pH 7.5 containing the same additives.
I1
=
Exchange reaction. The multisynthetase complex from rat liver (0.3 mg/ml, 0.2 pM) was incubated in 50 mM TridHCI, pH 7.5, 0.5 M KC1, 10 mM 2-mercaptoethanol in the presence of variable concentrations of rAspRSK-P,and rAspRS or AspRS AN20 where indicated. Incubation was conducted at 25°C for the times indicated. The mixture was then immediately applied to a Superose 6 HR 10/30 column (Pharmacia) developed at a flow rate of 0.4 ml/min at 20°C in the same buffer. Fractions of 0.5 ml were collected. The complex-associated and free forms of AspRS are eluted after 28 min and 38 min, respectively. The effective separation of the two forms during chromatography requires about 10 min, corresponding to the actual time to of the exchange reaction. Fractions containing the complex were combined and the incorporated radioactivity determined by scintillation counting. For each experiment, the global yield was determined by the absorbance at 280nm and by assaying IleRS or ArgRS activities, two components of the complex. Approximately 60% of the complex supplied in the incubation mixture was recovered in the high molecular-mass fractions. Analysis of exchange data. The exchange of the endogenous AspRS component of the multisynthetase complex (Cx AspRS) by the ”P-labelled recombinant free species rAspRSK-P was followed by measuring the formation of the Cx .rAspRSK-P species, corresponding to the amount of rAspRSK-Passociated to the complex. The kinetics of formation of the Cx.rAspRSK-P entity was consistent with a mechanism of exchange by dissociation, that can be written as follows: I
Cx.AspRS
+ rAspRSK-P
Cx
+ AspRS + rAspRSK-P + AspRS ,
2 Cx .rAspRSK-P
where Cx is an unstable intermediate corresponding to a complex deprived of its AspRS component. This two step mechanism was analyzed as the sum of four simultaneous reactions : CX. AspRS
Cx
- + - + k-i
CX
CX + AspRS
ki
+ rAspRSK-P
k?
Cx.rAspRSK-P
1
AspRS
CX.AspRS Cx.rAspRSK-P
2
Cx
rAspRSK-P
K , and K2 correspond to the respective dissociation constants Kd for AspRS ( k - , / k , ) and rAspRSK-P( k , / k 1 ) . At steady state, consider that K , = n K , , therefore:
The concentration of the radiolabelled, free AspRS and of the complex initially added can be expressed as: [rAspRSK-P],,= [rAspRSK-PI,, + [Cx .rAspRSK-PI,, [Cx.rAspRS],, = [Cx.rAspRS],, + [Cx.rAspRSK-Pl,, + [Cx],,,, where the concentration of the unstable intermediate [Cx],, is negligible. In addition, since [AspRS], = 0, [AspRS],,, can be approximated to [Cx .rAspRSK-P],,, Eqn (1) can be written:
[rA\pRSK-P],[Cx.rA
